The Molecular Evolution of Cetacean Dim- Light Vision: in Vitro Studies of Rhodopsin Over a Macroevolutionary Transition
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The Molecular Evolution of Cetacean Dim- Light Vision: In Vitro Studies of Rhodopsin Over a Macroevolutionary Transition by Sarah Dungan A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Ecology and Evolutionary Biology University of Toronto © Copyright by Sarah Dungan 2018 The Molecular Evolution of Cetacean Dim-Light Vision: In Vitro Studies of Rhodopsin Over a Macroevolutionary Transition Sarah Dungan Doctor of Philosophy Department of Ecology and Evolutionary Biology University of Toronto 2018 Abstract Cetaceans (whales and dolphins) are fully aquatic mammals that have captured the imagination of biologists due to their iconic evolutionary transformation from terrestrial ancestors. Nevertheless, much about how this extreme macroevolutionary transition occurred at the molecular level remains unknown. The vertebrate visual system is ideal for studying molecular adaptation due to the reliance organisms place on it for fitness-related behaviours, and because key molecular components of the visual transduction cascade in photoreceptors are relatively well understood. Light activation of the dim-light pigment, rhodopsin, is the first step of the visual transduction cascade, and is thought to have been the target of selection across a wide variety of vertebrate lineages. Cetaceans, due to their rich evolutionary history, are model organisms for understanding how changing environments put selective pressure on sensory processes. In this dissertation, I investigate the molecular basis for adaptive evolution in cetacean rhodopsin using a combination of in vitro expression experiments and computational analyses. First, I evaluate evidence for adaptation in cetacean rhodopsin through characterization of its spectral properties in the killer whale (Orcinus orca), and a statistical assessment of selection patterns in a representative dataset of cetacean rhodopsin genes. Next, I focus on the evolutionary significance of key amino acid substitutions in killer whale rhodopsin that confer both spectral and non-spectral (kinetic) functional shifts, specifically through comparison with rhodopsin pigments from two outgroup ii species (bovine and hippopotamus). Finally, I use an ancestral sequence reconstruction and protein resurrection approach to establish functional and corresponding molecular shifts across the terrestrial-aquatic transition at the root of Cetacea. Together, these projects provide a more complete investigation of cetacean rhodopsin, not only addressing questions regarding dim-light (aquatic) adaptation in cetaceans, but also generating new hypotheses about how the ecology of living and ancient cetaceans has shaped the visual system. iii Acknowledgements I would like to begin by acknowledging the land on which this research was conducted (University of Toronto, St. George campus). This land is the traditional territory of the Wendat, Anishnaabeg, Haudenosaunee, Métis, and Mississaugas of the New Credit First Nation. The St. George campus and surrounding city are still home to many indigenous peoples from across Turtle Island, and I am grateful to have had the opportunity to work on this land. My graduate studies were supported by a Natural Sciences and Engineering Research Council of Canada (NSERC) post-graduate scholarship, and a Vision Science Research Program (VSRP) fellowship. My supervisor, Belinda Chang, was supported by an NSERC Discovery Grant and a Human Frontier Science Program (HFSP) Grant. The 11-cis retinal chromophore used in visual pigment experiments was generously provided by Rosalie Crouch (Medical University of South Carolina). Many thanks go to my advisory committee members, David Irwin and Zhaolei Zhang, my appraisal committee members, Deborah McLennan and David Evans, and the EEB department faculty who have provided advice and mentorship over the years (particularly Helen Rodd and Maydianne Andrade). My appreciation also goes to all the staff members of both the EEB and CSB departments who keep everything running, especially Kitty Lam, Tamar Mamourian, and Jim Dix. I am also grateful for the friendship of my fellow EEB and Chang lab graduate students, Nihar Bhattacharyya, Frances Hauser, and Gianni Castiglione in particular. Without you, these last five years would have been filled with far more anxiety, and many fewer laughs. To my supervisor, Belinda Chang, thank you for taking a chance on a big whale dork with no wet lab experience. All the new skills I’ve learned in your lab, combined with my more whale-focused tendencies, have paid off with this interdisciplinary dissertation that I hope does the uniquely balanced position of your program between protein and organismal evolution justice. Finally, to my family: my parents, sister, and most especially my partner, Jessica Edwards. Thank you for putting up with me through all the anxiety, depression, and general angst. The mental health journey I’m taking has made me a stronger, wiser, more compassionate person, and I can’t wait to explore the future with you. iv Table of Contents Acknowledgements iv Table of Contents v List of Figures vii List of Tables viii Chapter 1: Introduction and literature review 1 1 - Studying molecular adaptation in natural systems 1 2 - Detecting signatures of selection in coding sequences 6 3 - An overview of mammalian visual pigments and phototransduction 9 4 - Cetaceans as models for molecular evolutionary study 13 5 - Thesis objectives 17 Chapter 2: Positive selection in cetacean rhodopsin 19 1 - Abstract 19 2 - Introduction 20 3 - Results 22 4 - Discussion 36 5 - Methods 41 Chapter 3: Intramolecular epistasis in cetacean rhodopsin 46 1 - Abstract 46 2 - Introduction 47 3 - Results 50 4 - Discussion 59 5 - Methods 64 Chapter 4: Ancestral cetacean rhodopsin reconstruction 66 1 - Abstract 66 2 - Introduction 67 3 - Results 70 v 4 - Discussion 81 5 - Methods 88 Chapter 5: Cetacean vision beyond rhodopsin 92 1 - Significance and future directions 92 2 - Opsin pseudogenization in cetaceans 94 3 - Systems-level sensory biology for cetaceans 96 References 98 Appendix I: Supplementary materials for chapter 2 114 Appendix II: Supplementary materials for chapter 3 124 Appendix III: Supplementary materials for chapter 4 133 vi List of Figures Chapter 2 Figure 2.1: Killer whale rhodopsin spectral tuning 24 Figure 2.2: Killer whale rhodopsin 3D structure 26 Figure 2.3: Cetacean species tree with ecological clade partitions 29 Figure 2.4: Site-by-site dN/dS across cetacean rhodopsin 34 Chapter 3 Figure 3.1: Spectral tuning and retinal release of killer whale and hippo rhodopsin 51 Figure 3.2: Epistasis in killer whale vs. bovine rhodopsin retinal release 55 Figure 3.3: Reconstruction of sites 83, 292, and 299 on the cetacean species tree 57 Figure 3.4: Killer whale metarhodopsin II 3D structure 58 Chapter 4 Figure 4.1: Ancestral cetacean rhodopsin 3D structure and snake plot 71 Figure 4.2: Functional shifts in rhodopsin over the Cetacea root branch 76 Figure 4.3: Backward mutations in ancestral cetacean rhodopsin spectral tuning 79 Figure 4.4: Backward mutations in ancestral cetacean rhodopsin retinal release 80 vii List of Tables Chapter 2 Table 2.1: Spectral tuning of killer whale and mutant rhodopsins 25 Table 2.2: Random-sites model statistics (PAML) 28 Table 2.3: Clade model statistics (PAML) 31 Table 2.4: Positively selected sites in cetacean rhodopsin 35 Chapter 3 Table 3.1: Retinal release in killer whale, bovine, and mutant rhodopsins 52 Chapter 4 Table 4.1: Summary of terrestrial-aquatic rhodopsin substitutions 74 Table 4.2: Spectral tuning and retinal release in ancestral rhodopsins 77 viii 1 Chapter 1 Introduction and literature review “One often hears of writers that rise and swell with their subject, though it may seem but an ordinary one. How, then, with me, writing of this Leviathan?” — Herman Melville 1 Studying molecular adaptation in natural systems Natural selection is foundational to organismal evolution (Darwin 1859), and genetic variation is the substrate on which it operates (Fisher 1930). These are fundamental truths of biology, yet the link between molecular diversity and organismal fitness remains difficult to both demonstrate and mechanistically reconstruct (Barrett and Hoekstra 2011). Functional and structural changes in proteins typically arise from non-synonymous substitutions: mutations in the gene coding sequence that alter amino acid identity (Kimura 1983). If these amino acid substitutions affect the protein phenotype such that traits linked to survival or reproductive success are improved, the molecular changes may eventually become fixed in a population. While associations between genes and adaptive phenotypes have grown increasingly common in the literature (reviewed in Stapley et al. 2010), relatively fewer studies extend the link to the level of individual mutations. As an exception, consider coat colour in wild populations of deer mice. Mice with lighter coats are less frequently caught by avian predators (Vignieri et al. 2010), and this phenotype has been mechanistically linked with the agouti gene locus (Linnen et al. 2009). Not only that, but explicit associations between agouti single-nucleotide polymorphisms (SNPs) and the light colour morph have also been determined from genome-wide datasets. These SNPs were under positive selection, with light colour alleles having significantly higher selection coefficients (Linnen et al. 2013). Structurally, the light colour alleles likely facilitate synthesis of pheomelanin (yellow pigment)